An improved understanding of the effects of steroid hormones on target cells is important for the treatment of hormone dependent breast tumors. While extensive work has been carried out on steroid receptors and their action in the nucleus in activating new gene transcription, the possibility that steroids also act on cells in other ways has received less attention. In the proposed studies we will focus, in part, on possible extra-nuclear actions of estrogens. We will use a human breast cancer cell line, MCF-7, which contains cytoplasmic receptors for estrogen, androgen, progestins and glucocorticoids, as a model system. These cells are stimulated by estrogens, androgens and insulin. We will study the early response of these cells to estrogen by measuring changes in the rate and type of RNA and protein synthesis, changes in the level of cAMP and cGMP and alterations in phospholipid metabolism. Changes in all of these areas have been reported in target tissues in vivo in response to estrogen. We will then enucleate cells using cytochalasin B and determine whether the enucleated cells are capable of any of the responses to estrogen treatment listed above. Mutants of MCF-7 with altered sensitivity to estrogen will be obtained by selecting clones of cells which are resistant to anti-estrogens such as Tamoxifen, which inhibit the growth of MCF-7 cells in culture. Mutant cells will be studied to determine what steps in their response to estrogen are blocked, including whether or not they still contain a receptor and whether it migrates to the nucleus in response to estrogen. We will concentrate upon obtaining temperature-sensitive mutants and will search in particular for a mutant with a temperature-sensitive receptor protein. We would use such a mutant by studying the effect on the cells' response to estrogen of inactivating the receptor at various times following estrogen stimulation. We will determine, in cultures of synchronized MCF-7 cells, whether the nature of the response to estrogen varies with the phase of the cell cycle. We will also use synchronized cells to determine when, during the cell cycle, the receptor protein is synthesized and when, following DNA synthesis, the new chromatin develops acceptor activity for the receptor.